Lipids are important regulators of the activity of many proteins including those involved in cardiac regulation, yet little is known about the molecular mechanisms mediating these effects. The overall aim of this proposal is to investigate the structural determinants of protein-lipid interactions, and to define how these interactions contribute to signal transduction in the cardiovascular system. We will use as a model the binding of an isozyme of inositol phospholipid-specific phospholipase C (PI-PLC), PLC delta 1, to its substrate, phosphatidylinositol 4,5- bisphosphate (PIP2). Our data and that from the literature suggest that a site of lipid interaction resides in a newly discovered protein module of 100 amino acids, termed the pleckstrin homology (PH) domain. This novel domain exists in many molecules (including PLC delta 1) which participate in signal transduction. Its biology and function while of great general interest, is currently poorly understood. We hypothesize that a major function of PH domains in general, and specifically for PLC delta 1, is to bind phospholipid. This hypothesis challenges the commonly held belief that PH domains primarily mediate protein-protein interactions. We further hypothesize that PH domains and more generally phospholipid interaction with proteins serve as important regulatory elements in cardiovascular and neural signal transduction. Specific Goals for the proposal include the following: 1. To characterize using molecular biological and biochemical techniques high affinity substrate (PIP2) and non-substrate (PS) binding to PLC delta 1. Mutagenesis will be performed to delineate the binding site. The stoichiometry of phospholipid binding will be determined. It will be determined if PS and PIP2 sites are equivalent. 2. To determine the mechanisms by which PS and high affinity binding of PIP2 activate PLC delta 1. Contributions of enzyme anchoring, processivity, and catalysis to activation by lipids will be assessed. 3. To investigate the nature of ligand specificity for the PH domains in PLC delta 1 and other molecules. A wide range of ligands and the generality of PH domain function will be investigated. These studies should not only increase understanding of basic cardiovascular regulation, but also have the potential to promote the development of novel post-receptor therapeutics and new diagnostic tools.